健康人和肝癌患者唾液糖蛋白糖链谱的研究
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摘要
目的：蛋白质糖基化是最重要的翻译后修饰之一,参与各种生命活动过程,近年研究发现在肝癌的发生发展过程中伴有糖蛋白糖链结构和功能的改变。唾液作为体液的一种,含有丰富的N-连接和0-连接糖蛋白,且相较于血液取材更为方便,对机体无损伤,因此从唾液中筛选生物标记物进行疾病早期诊断,己引起广泛关注。目前唾液与疾病关系的研究主要集中在疾病发生过程中唾液蛋白质的丰度和种类变化方面,唾液糖蛋白和糖蛋白糖链结构变化与癌症的相关性研究较少,因此本研究主要利用高通量、高灵敏性的凝集素芯片技术,检测不同年龄段健康人和肝癌患者唾液,分析比较不同年龄段健康人唾液糖蛋白糖链表达差异,以及肝癌患者唾液中特有的糖蛋白糖链,以期从唾液中筛选出特异性强的糖蛋白糖链作为肝癌诊断标志物。
方法：收集三个不同年龄段健康志愿者唾液样本和肝癌患者唾液样本,提取、纯化唾液蛋白质后,Cy3荧光标记,然后与凝集素芯片孵育,扫描获取芯片图像,GenePix3.0软件读取荧光信号值,对数据进行中值归一化和Raito值分析,比较不同年龄段不同性别健康人唾液糖蛋白糖链差异,筛选肝癌患者和健康人唾液中差异表达的糖蛋白糖链。
结果：1.应用凝集素芯片检测健康志愿者唾液,发现不同年龄段不同性别的健康人唾液糖蛋白糖链结构存在差异,其中：(1)健康儿童男性与女性之间唾液糖蛋白糖链表达差异较小,男性儿童唾液中Gal结构高表达,而女性儿童唾液中GalNAc、β-l,4GlcNAc和LacNAc结构高表达；(2)健康成年人男性与女性之间唾液糖蛋白糖链表达差异较为显著,其中末端GalNAc、Gal、β-l,4GlcNA、LacNAc和a-1,6 Man糖链结构在男性成年人中高表达,而女性成年人唾液中则是GlcNA、aGalNAc、aGal、GalNAcα-Ser/Thr(Tn)、核心Fuca-1,6、Fuca-1,2和Sia2-6Galβ1-4Glc(NAc)糖链结构表达量较高；(3)健康老年人男性唾液中的糖蛋白糖链结构相较于健康老年人女性唾液中更为丰富,其中Gal、αGalNAc、Manα-1,3和a-1,6连接、核心Fuca-1,6以及GalNAcα-Ser/Thr(Tn)糖链结构在男性老年人唾液中高表达,而女性唾液中仅有Fuca-1,3、末端GalNAc和唾液酸化的路易斯聚糖抗原结构(sLex)高表达。
2.通过凝集素芯片分别检测7例肝癌患者(老年人男性)唾液和同年龄段健康男性老年人唾液的初步研究发现：在肝癌患者唾液中,凝集素RCA12O、WFA、PHA-E、AAL、LTL和SNA识别的Gal、GalNAc、平分型GlcNAc、双天线型N-糖链、Fuca-1,3、核心Fuca-1,6、唾液酸化的路易斯聚糖抗原结构(sLex)、Siaa-2,6糖链结构高表达；而凝集素BS-Ⅰ、PTL-Ⅱ、DSA. SBA、ConA、GNA、GSL-Ⅰ、VVA、MAL-Ⅱ和WGA识别的αGalNAc、αGal、β1-4GlcNAc、末端GalNAc、αMan、GalNAcα-Ser/Thr(Tn)以及Siaa-2,3连接和多价Sia糖链结构在肝癌患者唾液中低表达。
结论：1.健康人唾液中N-连接和O-连接糖蛋白丰富多样。随着年龄增长,健康人唾液中糖蛋白糖链增多,而且同年龄段不同性别健康人唾液糖蛋白糖链结构存在表达差异,其中成年人男性与女性唾液糖蛋白糖链表达差异最为显著。
2.肝癌患者唾液中Gal、GalNAc、平分型GlcNAc、双天线型N-糖链、Fucα-1,3、核心Fucα-1,6、Siaα-2,6和sialyl-Lewisx抗原结构高表达,提示这些糖链可能作为肝癌诊断的潜在标志物。
Purpose：Glycosylation is an important component for a number of biological processes and is the most abundant and complicated of the known post-translational modifications found on proteins. The occurrences and developments of liver cancer are accompanied by changes in structures and functions of glycoproteins. Saliva, as a body fluid, is a rich source of N-and O-linked glycoproteins. Saliva testing, a simple and non-invasive alternative to serum, has drawn widely attention of researchers to find saliva biomarker for monitoring physiological and pathological conditions in humans, as well as for early diagnosis of diseases. At present, researches on the relationship between saliva and disease mainly focus in the changes of salivary proteins abundance and kinds, and glycoprofiling of the human salivary associated with the processes of cancer are very little involved. Therefore this study analyzed human salivary glycoproteins by high-throughput and sensitivity lectin microarrys, then identifyed and compared the glycopatterns of healthy people associated with different ages groups and different gender. And we expect to find specific saliva biomarkers from liver cancer patients for early diagnosis of liver cancer.
Method:First, saliva samples were collected from healthy people of three different age groups and liver cancer patients; then extracted and purifyed salivary proteins, labled them with Cy3 fluorescent dye and then incubated with lectin microarray. According to the principle of lectin to glycan binding affinity, we analyzed the salivary glycoproteins differences of healthy people associated with different gender in different age groups, and screened differential expression glycopatterns between liver cancer patients and healthy people.
Results:1. By analyzing healthy people saliva, we found that kinds of glycoproteins make up a major and important part of human salivary proteome, including:(1) There were little glycopattern expression differences between male and female children, Galactose was higher in male children, while GalNAc、β-1,4 GlcNAc and LacNAcwere higher in female children; (2) The differences of salivary glycan structures between healthy male and female adults were significantly, terminal GalNAc、Gal、β-1,4GlcNAc、LacNAc andα-1,6Man structures were more expressed in male adults; while GlcNAc、αGalNAc、αGal、GalNAcα-Ser/Thr(Tn)、Sia2-6Gαlβ1-4Glc(NAc), Fucα-1,2and core Fucα-1,6 structures were more expressed in female adults; (3) Salivary glycoproteins glycan structures were more abundant in healthy older men than in women. In the older men saliva, Gal、αGalNAc、α-1,3 andα-1,6 linked mannose、core Fucα-1,6 and GalNAcα-Ser/Thr(Tn-antigen) structures were induced, whereas Fucα-1,3、terminal GalNAc andsialyl-Lewisx(sLex)structures were higher in older women salivary glycoproteins.
2. Seven salivary samples of liver cancer patients(older men) and healthy people in the same age group and gender were initially analyzed by lectin microarrays. The results were as follows:in liver cancer patients, Gal、GalNAc bisecting GlcNAc、biantennary N-glycans、Fucα-1,3、core Fucα-1,6、sialyl-Lewisx(sLex) and Siaα-2,6 glycan structures which specificitily recognized by RCA120、WFA、PHA-E、AAL LTL and SNA were increased; whereasαGalNAc、αGal、β1-4GlcNAc、terminal GalNAc、αMan、GalNAcα-Ser/Thr(Tn)、Siaα-2,3and mμltivalent Sia glycan structures that recognized by BS-I、PTL-Ⅱ, DSA、SBA、ConA、GNA, GSL-ⅠVVA、MAL-Ⅱand WGA were reduced in liver cancer patients.
Conclusion:1. Saliva is a rich source of N-and O-linked glycoproteins. With the growth of age, salivary glycoproteins of healthy people is increasing, and glycopattern expression of different gender in the same age group is differed. The salivary glycan structures differences between healthy male and female adults were the most significantly.2. Gal、GalNAc、bisecting GlcNAc、biantennary N-glycans、Fucα-1,3、core Fuca-1,6、sialyl-Lewisx(sLex) and Siaα-2,6 glycan structures are higher in liver cancer patients, suggesting that these salivary glycopatterns may be used as a potential diagnostic biomarker of liver cancer.
方法：收集三个不同年龄段健康志愿者唾液样本和肝癌患者唾液样本,提取、纯化唾液蛋白质后,Cy3荧光标记,然后与凝集素芯片孵育,扫描获取芯片图像,GenePix3.0软件读取荧光信号值,对数据进行中值归一化和Raito值分析,比较不同年龄段不同性别健康人唾液糖蛋白糖链差异,筛选肝癌患者和健康人唾液中差异表达的糖蛋白糖链。
结果：1.应用凝集素芯片检测健康志愿者唾液,发现不同年龄段不同性别的健康人唾液糖蛋白糖链结构存在差异,其中：(1)健康儿童男性与女性之间唾液糖蛋白糖链表达差异较小,男性儿童唾液中Gal结构高表达,而女性儿童唾液中GalNAc、β-l,4GlcNAc和LacNAc结构高表达；(2)健康成年人男性与女性之间唾液糖蛋白糖链表达差异较为显著,其中末端GalNAc、Gal、β-l,4GlcNA、LacNAc和a-1,6 Man糖链结构在男性成年人中高表达,而女性成年人唾液中则是GlcNA、aGalNAc、aGal、GalNAcα-Ser/Thr(Tn)、核心Fuca-1,6、Fuca-1,2和Sia2-6Galβ1-4Glc(NAc)糖链结构表达量较高；(3)健康老年人男性唾液中的糖蛋白糖链结构相较于健康老年人女性唾液中更为丰富,其中Gal、αGalNAc、Manα-1,3和a-1,6连接、核心Fuca-1,6以及GalNAcα-Ser/Thr(Tn)糖链结构在男性老年人唾液中高表达,而女性唾液中仅有Fuca-1,3、末端GalNAc和唾液酸化的路易斯聚糖抗原结构(sLex)高表达。
2.通过凝集素芯片分别检测7例肝癌患者(老年人男性)唾液和同年龄段健康男性老年人唾液的初步研究发现：在肝癌患者唾液中,凝集素RCA12O、WFA、PHA-E、AAL、LTL和SNA识别的Gal、GalNAc、平分型GlcNAc、双天线型N-糖链、Fuca-1,3、核心Fuca-1,6、唾液酸化的路易斯聚糖抗原结构(sLex)、Siaa-2,6糖链结构高表达；而凝集素BS-Ⅰ、PTL-Ⅱ、DSA. SBA、ConA、GNA、GSL-Ⅰ、VVA、MAL-Ⅱ和WGA识别的αGalNAc、αGal、β1-4GlcNAc、末端GalNAc、αMan、GalNAcα-Ser/Thr(Tn)以及Siaa-2,3连接和多价Sia糖链结构在肝癌患者唾液中低表达。
结论：1.健康人唾液中N-连接和O-连接糖蛋白丰富多样。随着年龄增长,健康人唾液中糖蛋白糖链增多,而且同年龄段不同性别健康人唾液糖蛋白糖链结构存在表达差异,其中成年人男性与女性唾液糖蛋白糖链表达差异最为显著。
2.肝癌患者唾液中Gal、GalNAc、平分型GlcNAc、双天线型N-糖链、Fucα-1,3、核心Fucα-1,6、Siaα-2,6和sialyl-Lewisx抗原结构高表达,提示这些糖链可能作为肝癌诊断的潜在标志物。
Purpose：Glycosylation is an important component for a number of biological processes and is the most abundant and complicated of the known post-translational modifications found on proteins. The occurrences and developments of liver cancer are accompanied by changes in structures and functions of glycoproteins. Saliva, as a body fluid, is a rich source of N-and O-linked glycoproteins. Saliva testing, a simple and non-invasive alternative to serum, has drawn widely attention of researchers to find saliva biomarker for monitoring physiological and pathological conditions in humans, as well as for early diagnosis of diseases. At present, researches on the relationship between saliva and disease mainly focus in the changes of salivary proteins abundance and kinds, and glycoprofiling of the human salivary associated with the processes of cancer are very little involved. Therefore this study analyzed human salivary glycoproteins by high-throughput and sensitivity lectin microarrys, then identifyed and compared the glycopatterns of healthy people associated with different ages groups and different gender. And we expect to find specific saliva biomarkers from liver cancer patients for early diagnosis of liver cancer.
Method:First, saliva samples were collected from healthy people of three different age groups and liver cancer patients; then extracted and purifyed salivary proteins, labled them with Cy3 fluorescent dye and then incubated with lectin microarray. According to the principle of lectin to glycan binding affinity, we analyzed the salivary glycoproteins differences of healthy people associated with different gender in different age groups, and screened differential expression glycopatterns between liver cancer patients and healthy people.
Results:1. By analyzing healthy people saliva, we found that kinds of glycoproteins make up a major and important part of human salivary proteome, including:(1) There were little glycopattern expression differences between male and female children, Galactose was higher in male children, while GalNAc、β-1,4 GlcNAc and LacNAcwere higher in female children; (2) The differences of salivary glycan structures between healthy male and female adults were significantly, terminal GalNAc、Gal、β-1,4GlcNAc、LacNAc andα-1,6Man structures were more expressed in male adults; while GlcNAc、αGalNAc、αGal、GalNAcα-Ser/Thr(Tn)、Sia2-6Gαlβ1-4Glc(NAc), Fucα-1,2and core Fucα-1,6 structures were more expressed in female adults; (3) Salivary glycoproteins glycan structures were more abundant in healthy older men than in women. In the older men saliva, Gal、αGalNAc、α-1,3 andα-1,6 linked mannose、core Fucα-1,6 and GalNAcα-Ser/Thr(Tn-antigen) structures were induced, whereas Fucα-1,3、terminal GalNAc andsialyl-Lewisx(sLex)structures were higher in older women salivary glycoproteins.
2. Seven salivary samples of liver cancer patients(older men) and healthy people in the same age group and gender were initially analyzed by lectin microarrays. The results were as follows:in liver cancer patients, Gal、GalNAc bisecting GlcNAc、biantennary N-glycans、Fucα-1,3、core Fucα-1,6、sialyl-Lewisx(sLex) and Siaα-2,6 glycan structures which specificitily recognized by RCA120、WFA、PHA-E、AAL LTL and SNA were increased; whereasαGalNAc、αGal、β1-4GlcNAc、terminal GalNAc、αMan、GalNAcα-Ser/Thr(Tn)、Siaα-2,3and mμltivalent Sia glycan structures that recognized by BS-I、PTL-Ⅱ, DSA、SBA、ConA、GNA, GSL-ⅠVVA、MAL-Ⅱand WGA were reduced in liver cancer patients.
Conclusion:1. Saliva is a rich source of N-and O-linked glycoproteins. With the growth of age, salivary glycoproteins of healthy people is increasing, and glycopattern expression of different gender in the same age group is differed. The salivary glycan structures differences between healthy male and female adults were the most significantly.2. Gal、GalNAc、bisecting GlcNAc、biantennary N-glycans、Fucα-1,3、core Fuca-1,6、sialyl-Lewisx(sLex) and Siaα-2,6 glycan structures are higher in liver cancer patients, suggesting that these salivary glycopatterns may be used as a potential diagnostic biomarker of liver cancer.
引文
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